This invention relates to liquid chromatography (LC) and mass spectrometry (MS). More particularly, this invention is concerned with both a method and apparatus for providing improved creation and detection of ions by use of photoionization (PI), in conjunction with LC and MS.
While atmospheric pressure photoionization (APPI) is known, it has not previously been applied to liquid chromatography-mass spectrometry (LC-MS). Furthermore, there have been very few reports of PI combined with LC, despite the longstanding use of photoionization detection (PID) with gas chromatography (GC).
Photoionization detection in GC typically involves the use of a discharge lamp that generates vacuum-ultraviolet (VUV) photons. If one of these photons is absorbed by a molecule in the column eluant with a first ionization potential (IP) lower than the photon energy, then single photon ionization may occur. The photoions thereby generated are detected as current flowing through a suitable collection electrode; a chromatogram can be obtained by plotting the current detected during a chromatographic run versus time. For PID-GC, the discharge lamp is normally selected such that the energy of the photons is greater than the IP of the analyte, but below the IP of the carrier gas. (Most organic molecules have ionization potentials in the range of 7-10 eV; the common GC carrier gases have higher values, e.g. helium, 23 eV). Ionization of the analyte can then occur selectively and low background currents may be achieved.
There are a few earlier reports in the literature of combining LC and PI. (Schermund, J. T., Locke, D. C. Anal. Lett. 1975, 8, 611-625; Locke, D. C., Dhingra, B. S., Baker, A. D. Anal. Chem. 1982, 54, 447-450; Driscoll, J. N., Conron, D. W., Ferioli, P., Krull, I. S., Xie, K.-H. J. Chromatogr. 1984, 302, 43-50; De Wit, J. S. M., Jorgenson, J. W. J. Chromatogr. 1987, 411, 201-212). However, these also relied upon direct detection of the photoion current, without mass analysis. Selective ionization was possible in these experiments, too, because the common LC solvents also have relatively high IP""s (water, IP=12.6 eV; methanol, IP=10.8 eV; acetonitrile, IP=12.2 eV). Thus, these methods were similar to photoionization detection as used with GC. In the majority of cases the liquid eluant from the LC column was completely vaporized before it entered the ionization region, and ionization took place in the vapour phase. However, one of these studies involved direct photoionization of the liquid-phase eluant (Locke, D. C., Dhingra, B. S., Baker, A. D. Anal. Chem. 1982, 54, 447-450.)
When trace levels of analyte must be detected in the presence of a great excess of carrier gas or solvent, and ion current alone is being measured, it is essential that photoionization be selective. Otherwise, ions generated from the carrier gas or solvent could overwhelm the analyte ions of interest. However, this requirement may be obviated if a mass analyzer is used to separate the photoions prior to detection, i.e. so as to separate desired analyte ions from other ionized species, such as those arising from solvent molecules or any impurities.
There is also a small number of reports of APPI combined with mass spectrometry. The inventors are aware of only three reports of true mass analysis of photoions created at atmospheric pressure (Revel""skii, I. A.; Yashin, Vosnesenskii, V. N.; Y. S.; Kurochkin, V. K.; Kostyanovksii, R. G.; Izv. Akad. Nauk SSSR, Ser. Khim. 1986, (9) pp. 1987-1992; Revel""skii, I. A.; Yashin, Y. S.; Kurochkin, V. K.; Kostyanovksii, R. G.; Chemical and Physical Methods of Analysis 1991, 243-248 translated from Zavodskaya Laboratoiya 1991, 57, 1-4; Revel""skii, I. A.; Yashin, Y. S.; Voznesenskii, V. N.; Kurochkin, V. K.; Kostyanovksii, R. G. USSR Inventor""s certificate 1159412, 1985), although there have been numerous examples of APPI coupled with ion mobility spectrometry (IMS) (Baim, M. A., Eatherton, R. L., Hill Jr., H. H. Anal. Chem. 1983, 55, 1761-1766; Leasure, C. S., Fleischer, M. E., Anderson, G. K., Eiceman, G. A. Anal. Chem. 1986, 58, 2142-2147; Spangler, G. E., Roehl, J. E., Patel, G. B., Dorman, A., U.S. Pat. No. 5,338,931, 1994; Doering, H.-R.; Arnold, G.; Adler, J.; Roebel. T.; Riemenschneider, J.; U.S. Pat. No. 5,968,837, 1999). In the three papers describing APPI-MS experiments that established the feasibility of the combination, direct analysis was performed of a gaseous mixture of samples in a flow of helium carrier gas. A hydrogen discharge lamp (hn=10.2 eV) was utilized to create ions from the gaseous mixture for analysis by a quadrupole mass spectrometer. Significantly, the relative abundance of sample ions in the spectra obtained of the sample mixture was found to depend upon sample concentration. At high sample concentrations, ion-molecule reactions, particularly charge (electron) transfer, distorted the appearance of the mass spectra: this charge transfer caused the majority of charge to be transferred to the species with the lowest IP. Another finding was that predominantly molecular or quasi-molecular ions are created by PI at atmospheric pressure, indicating that little fragmentation occurs during the ionization step. Finally, when solvent vapour (water or methanol) was introduced into the sample mixture carried in the helium stream, a decrease in sensitivity for the method was observed.
With regard to the prospect of combining APPI with LC-MS, the finding that the presence of solvent vapour decreases the efficiency of ion formation is troublesome. This effect was known to the last researchers to study PID-LC, who described how vaporized solvent molecules absorb the photons, thereby decreasing the flux available to create photoions from the sample (De Wit, J. S. M., Jorgenson, J. W. J. Chromatogr. 1987, 411, 201-212). Another interesting observation from the early APPI-MS studies is the effect that charge-transfer reactions have on the final appearance of the spectra. This observation tells of the fact that the relative abundance of ions in an APPI spectrum will depend upon the reactions that the original photoions undergo prior to mass analysis. As is generally true for atmospheric pressure ionization methods, the high collision frequency insures that species with high proton affinities and/or low ionization potentials tend to dominate the positive ion spectra acquired, unless special measures are taken to sample the ions from the source before significant reactions occur. (In the case of negative ion atmospheric pressure ionization, molecules with high gas phase acidity or high electron affinity dominate the negative ion spectra.)
Many conventional LC-MS instruments rely on a corona discharge to promote ionization. A common configuration provides a heated nebulizer, known to those skilled in the art, for nebulization and vaporization of a sample solution, with the sample being introduced subsequent to a liquid chromatography step. The sample may also be introduced subsequent to a different liquid phase separation method, or from a liquid feeding device not involving a separation step (see the discussion of the preferred embodiment below).
A corona discharge (CD) has its own unique requirements. In the CD source, a high potential is necessary to create and maintain the discharge, which imposes restrictions on the use of separate ion transport mechanisms. A tube cannot be used to transport ions from the CD, because in order for a transport tube to have any effect it must be in close proximity to the ion source; in fact, it must enclose it. However, in order for the CD source to function, a strong electric field must be present at the needle tip, and if this field is maintained by applying the potential between the needle and the transport tube, then the ions produced will be quickly lost to the tube, due to the acceleration from the electric field; conversely, if the tube is held at a potential close to that of the needle, then ion loss from the above mechanism will be minimized, but few ions will be created, because of the lack of a suitably high field around the needle.
APCI can also be initiated by high energy electrons emitted from a radioactive 63Ni foil placed inside a narrow tube in an arrangement similar to the electron capture detector for GC. A 63Ni foil was successfully used in one of the early applications of atmospheric pressure ionization-mass spectrometry as a detector for LC (Horning, E. C., Carroll, D. I., Dzidic, I., Haegele, K. D., Horning, M. G., Stillwell, R. N., J. Chromatogr. Science 1974, 12, 725-729). However, a serious practical disadvantage of a 63Ni foil is the need for compliance with precautions and legal regulations concerning radioactive material.
No such restrictions are present in the APPI source, because the ionization is independent of the potential that the device is maintained at, and no radioactive materials are employed. This allows the position and shape of the transport tube to be selected without regard to maintaining a stable discharge (a further limiting factor of the CD source). Moreover, the potential on the tube can be controlled independently to optimize the transport of ions towards the sampling orifice. An additional electrostatic ion focussing element, or elements, may also be added to the ion source without affecting the ionization process, a unique feature of APPI (this is not practical for corona discharge or electrospray ionization).
For APPI, ion-molecule reactions occur in the transport tube between the dopant photoions, solvent molecules, and analyte molecules, with the net result being that charge is transferred to the analyte molecules (when favourable thermodynamic conditions exist).
The idea of using a dopant to increase the efficiency of ion formation by APPI is not entirely without precedent, as there have been several reported instances where dopants have been used with atmospheric pressure ionization. For instance, the use of acetone and toluene as dopants to enhance the sensitivity of PI-IMS has been described in patent application (WO 93/22033) and in U.S. Pat. No. 5,968,837. Also, charge-exchange reactions involving benzene have been successfully exploited to increase the sensitivity of corona discharge ionization towards samples with low proton affinity (Ketkar, S. N., Dulak, J. G., Dheandhanoo, S., Fite, W. L. Anal. Chim. Acta. 1991, 245, 267-270). To the inventors"" knowledge, a dopant has never before been used to enhance the production of photoions from the eluant of a liquid chromatograph.
What the present inventors have realized is that, while post-ionization reactions may complicate the analysis of APPI mass spectra, these reactions can be exploited to provide enhanced sensitivity. Where PI of vaporized LC eluants is undertaken, as described above, the direct PI of an analyte molecule is a statistically unlikely event, because of the excess of solvent molecules that may also absorb the limited photon flux. The lamps used to date for PI-LC have all had photon energies below the IP""s of the most commonly used LC solvents. This does substantially prevent ionization of the solvent, but nonetheless the solvent still absorbs the radiation preventing ionization of the desired analyte. Hence, the total ion production in these experiments has been quite low.
The present inventors have additionally realized that the number of ions produced by a discharge lamp can be greatly increased if the percentage of ionizable molecules in the vaporized LC eluant is raised to a significant fraction of the total. There are two means by which this can be achieved: 1) use a higher energy discharge lamp, so that the solvent molecules themselves are ionized; and, 2) add a large quantity of a dopant, having an IP below the photon energy, to the liquid eluant, or to the vapour generated from the eluant. If the recombination energy of the selected ionizable molecule is relatively high, or if its proton affinity is low, then the photoions of this molecule may react by proton or charge transfer with species present in the ionization region. For negative analyte ion formation, other mechanisms may be responsible, among others resonance electron capture, dissociative electron capture, ion pair formation, proton transfer and electron transfer. Because the ionization region is at atmospheric pressure, the high collision rate will ensure that the charge on the photoions is efficiently transferred to the analyte, provided that the thermodynamics are favourable. (Clearly, any number of competing reactions may also occur, depending upon the impurities present in the reaction region.)
There is a practical problem with using the first method (1) described above for increasing ion production, and that is the present lack of a window material that is both transparent to the requisite high energy photons, and stable in the presence of water. Also, the use of a higher energy lamp is necessarily accompanied by a loss of selectivity in ionization. For many applications, though, high selectivity is not desirable, because in case of unknown sample components, a universal, nonselective ionization method is desired. The present invention envisages exciting the solvent itself by using a suitable lamp. The benefit of the second method, (2) above, apart from the stability of the lamp window, is that the initial reagent ions can be selected; this is still possible with (1), but with fewer possibilities.
Additionally, the present invention can employ all lamp types for PI, pulsed as well as continuous output; the preferred embodiment utilizes a continuous lamp. The PI is then applied to LC (all liquid sample methods, whether or not separation is involved), with any suitable mass analyzer (triple-quadrupole, single-quadrupole, TOF, quadrupole-TOF, quadrupole ion trap, FT-ICR, sector, etc.).
Hence, possible mechanisms of ionization include: direct PI of vaporized analyte, ionization by ion-molecule reactions following PI of dopant in eluant, ionization by ion-molecule reactions following PI of solvent where the solvent acts as a dopant, etc. It does not matter which lamp is used for any of these, provided that the lamp""s energy is sufficient to ionize at least one major component of the eluant, or of the vapour generated from the eluant (the dopant can be introduced separately as a gas).
Windows made of lithium fluoride are optically transparent up to around 11.8 eV, and are used for argon lamps that can provide photons of 11.2, 11.6, and 11.8 eV (depending upon the lamp design). However lithium fluoride is hygroscopic, and these windows deteriorate quickly when exposed to moisture, a problem exacerbated by elevated temperatures. Consequently, due to the high water content in most LC solvent systems, and the high temperature required to vaporize the solvent, a lamp equipped with a lithium fluoride window may be expected to have only a limited useful lifetime. Nevertheless, it is conceivable that an argon discharge lamp could be used as a photoionization source for LC, but, if in the absence of a dopant, only if a major component of the solvent (e.g. methanol, ethanol, or iso-propanol) is ionizable by the lamp, and then only if special precautions are taken to protect the lamp""s window. An argon lamp can also be used in the manner of method (2), where no major component of the solvent itself is ionizable by the lamp, but a dopant is added. It should also be recognized that new window materials may become available, which would overcome the limitations of present lithium fluoride windows. Also, PI will conceivably work with windowless light sources if these become available.
The second method described above for enhancing ion production by APPI can eliminate the requirement for a lamp with a lithium fluoride window, by choosing a dopant species with a lower IP, so a different light source can be used. For example, for a dopant ionizable by 10 eV photons that has a suitably high recombination energy or low proton affinity, then a krypton discharge lamp may be used. Krypton lamps are usually equipped with magnesium fluoride windows that are much more stable in the presence of water vapour, and are optically transparent up to 11.3 eV. With a krypton lamp, it is possible to selectively ionize a dopant in the presence of solvent molecules, which provides the opportunity to gain some control over the ion-molecule chemistry in the ion source. The selectivity offered by this approach, along with the longer lifetimes anticipated for lamps equipped with magnesium fluoride windows, make the use of a dopant in combination with a lamp with a magnesium fluoride window the preferred method of implementing APPI in conjunction with LC-MS.
Lamps filled with argon or krypton are commercially available and are given as examples in the discussion above; lamps filled with other gases, producing the desired photon energies may be used equally well.
An advantage of the method of the present invention is that the sensitivity does not depend greatly on lamp current, which is inversely related to lamp lifetime; i.e., the lamp can be run at low powers without a great sensitivity drop (perhaps 10-15% difference in sensitivity between 0.4 mA and 2 mA). Consequently, the method provides the unanticipated benefit of being relatively economical. Without a dopant, sensitivity is proportional to lamp current; the mechanism responsible for the difference is as yet undetermined.
It is envisaged that irradiation of the sample will usually take place in the vapour phase, and that this will be the most efficient technique for most samples. However, it is possible to photoionize the liquid (Locke, D. C., Dhingra, B. S., Baker, A. D. Anal. Chem. 1982, 54, 447-450) before nebulization and vaporization. There are several factors to consider: 1) liquid phase solvent molecules have lower IP""s than isolated gas phase solvent molecules, and direct PI of most solvents will result with 10 eV photons; hence, a LiF window is not required; 2) Ion-electron recombination is much faster in the liquid phase so sensitivity will likely suffer; 3) direct contact between liquid and lamp window may hasten the rate of window deterioration. Based upon these factors, the method of the present invention can conceivably be applied in a manner either utilizing direct PI of liquids, followed by nebulization and vaporization, or utilizing PI of droplets created by nebulization, followed by vaporization. During the vaporization step, ions can be liberated from droplets in some arrangement similar to that utilized in the SCIEX TurbolonSpray ion source. However, the inventors do not believe that it would work as well as the preferred embodiments of the invention, as described below.
In accordance with a first aspect of the present invention, there is provided a method of analyzing a sample of an analyte, the method comprising:
(1) providing a sample solution comprising a solvent and an analyte as a sample stream;
(2) providing a dopant in the sample stream;
(3) forming a spray of droplets of the sample stream, to promote vaporization of the solvent and the analyte;
(4) vaporizing the droplets in said spray whereby the sample enters the vapour state;
(5) after step (2), in a region at atmospheric pressure, irradiating the sample stream with radiation to ionize the dopant, whereby at least one of subsequent collisions between said ionized dopant, and said analyte and indirect collisions of said analyte with solvent molecules acting as intermediates, results in ionization of said analyte; and
(6) passing the ions into the mass analyzer of a mass spectrometer for mass analysis of the ions.
The method can include, in step (5), irradiating the sample stream before step (4), to effect irradiation in the liquid state, or alternatively, irradiating the sample stream after step (4), to effect irradiation in the vapour state.
The step (2) of providing a dopant can comprise one of adding a separate dopant and utilizing the solvent as the dopant and the dopant can provided in one of the liquid state and the vapour state.
The method preferably includes providing a guide for guiding the sample stream in steps (3), (4) and (5), and this can be provided with an end shaped to promote focusing of the ions.
The method can include providing additional electrostatic focusing elements and a potential between a zone where the sample stream is irradiated in step (5) and the inlet of the mass spectrometer.
It is believed to be preferable to cause the sample stream to flow in a first direction in steps (3), (4) and (5), and in step (6) to pass the ions into a mass analyzer in a second direction, generally orthogonal to the first direction. However, the method also includes passing the sample stream in essentially the same direction in all of steps (3), (4), (5) and (6).
The method can be used to form either positive ions or negative ions in step (5).
The method can be effected on a sample solution including a plurality of analytes whereby all of said analytes are ionized to at least some extent, the method further including subjecting the analyte ions to a mass spectrometry step, to separate and to distinguish the different analytes.
The method can be effected on a sample solution which includes, prior to step (3), subjecting the sample stream to liquid phase separation, to separate said analyte from other substances.
Another aspect of the present invention provides an apparatus, for irradiation of a sample stream, formed from a sample solution including a relatively large amount of some ionizable species and a relatively small amount of an analyte to be ionized, the apparatus comprising:
spray means for forming a spray of droplets from the sample stream for vaporisation of the sample stream;
dopant supply means for supplying dopant to the sample stream; and
a means for irradiating the sample stream in a region at atmospheric pressure, to ionize the ionizable species at atmospheric pressure whereby at least one of: subsequent collisions between said ionized species and the analyte; and intermediate reactions between the ionized species and the analyte, results in charge transfer and ionization of the analyte; and
a mass spectrometer for determining the mass-to-charge ratio of the ions formed by irradiating the sample stream.
Preferably, the means for irradiation comprises a lamp, selected to provide photons having energy sufficient to ionize the ionizable species.
It is possible for the means for irradiating to comprise a laser.